1. Field of the Invention
The field of this invention relates to novel particles useful for detecting a plurality of specific binding interactions, especially the interactions in which at least one member is a biological molecule.
2. Background
Sensitive and specific methods are needed for quantitative analysis of trace amounts (10−8-10−10 M) of analytes in a sample. Direct measurements are usually difficult or impossible, because either the analyte does not possess a physical or chemical property (e.g., fluorescence) that is easily measurable, or because the sample contains interfering substances. A common approach to overcome this difficulty relies on the formation of a specific binding pair between an analyte of interest and a second member of a binding pair, with an easily detectable label linked to the second member of the binding pair. Specific binding pairs most commonly employed comprise antibody-antigen or antibody-hapten, ligand-receptor, lectin-sugar, avidin-biotin, DNA-DNA, DNA-RNA and RNA-RNA, where the nucleic acids may be of natural or synthetic origin.
There exist many assay formats that take advantage of a high specificity of the formation of properly selected binding pairs, including ELISA, RIA, immunoblotting, immunochromatographic, Southern blotting, Northern blotting, affinity chromatography and affinity electrophoresis. Such assays are usually carried out with the aim of detecting the presence and concentration of just one analyte or a small number of analytes. High throughput methods that rely on the formation of many binding pairs of one type such as DNA-DNA, having the capability of simultaneously detect hundreds, or thousands, of analytes are also known. Thus, DNA microarrays may contain tens of thousands of short oligonucleotides that are immobilized on a solid surface and are able to hybridize with tens of thousands of DNA fragments of complementary sequences suspected to be present in a sample. Current antibody arrays are suitable for the analysis of hundreds of antigens in a sample. While the DNA and protein microarrays have greatly increased our ability to analyze multiple analytes in biological samples, one of their drawbacks is the inherent difficulty to perform quality control of the individual microarrays. Other high throughput methods exist as well. Thus, an alternative to an array of spots characteristic for DNA and protein microarrays is an array of beads (U.S. Pat. No. 6,654,505 to Bridgham et al. and U.S. Pat. No. 6,355,431 to Chee et al.). A fiber optics device can be used with a bead array instead of a scanner or a CCD device usually employed for detecting the formation of specific binding pairs of DNA and protein micrarrays. Another method relies on the formation of specific binding pairs in a solution on the surface, of dispersed beads that are coded with various fluorescent dye combinations, so that each bead can be identified on the basis of its unique dye composition (U.S. Pat. No. 6,514,295 to Chandler et al.). Analytes are detected and quantified based on the fluorescence emitted by another dye linked to a second member that has participated in the formation of a specific binding pair at the surface of the color coded beads.
3. Description of Related Art
Electrophoretic methods have also been employed for detecting the formation of specific binding pairs. Thus, U.S. Pat. No. 5,084,150 to Karger et al. discloses a method combining electrophoresis and chromatography wherein charged colloidal particles selectively interact with analytes through affinity groups attached to the surface of the particles. The analytes distribute themselves between the electrophoresis medium and particles, so that migration rates of the analytes are determined by the equilibrium constants and the migration rate of the colloidal particles. U.S. Pat. No. 5,137,609 to Manian et al. discloses a differential separation assay wherein an analyte is reacted with excess of a fluorescently labeled second member of a binding pair, followed by the detection of the free and bound second member using pre-calibrated migration times of the two species.
U.S. Pat. No. 5,536,382 to Sunzeri discloses a capillary electrophoresis method for detecting an analyte is a clinical sample, based on electrophoretic separation of the unbound from bound analyte followed by their detection. Bao et al. in U.S. Pat. No. 5,810,985 disclose an electrochemically mediated chemical analysis wherein an analyte is introduced into a capillary containing a reagent whose contact with the analyte results in forming, or breaking, of a covalent bond. As either the analyte or the reagent is charged, the formation or breaking of a covalent bond causes a change in electrophoretic mobility of the analyte. U.S. Pat. No. 5,958,202 to Regnier et al. discloses a capillary electrophoresis enzyme immunoassay wherein a competitor mediates either the formation or depletion of a detectable product in a capillary subjected to an electric potential. In U.S. Pat. No. 5,630,924 Fuchs et al. disclose a method for analyte detection based on the formation of a three-member complex, including a first binding member carrying a detectable label, the analyte, and a second binding member comprising a charge-modifying moiety. During electrophoresis, the three-member complex is separated from the free first member that carries the label. In U.S. Pat. No. 6,673,550 Matray et al. disclose electrophoretic tag (e-tag) reagents comprising fluorescent compounds and mobility modifiers, wherein after the formation of specific binding pairs the tags are released by cleaving a cleavable linkage by oxidation. The mobility modifier has from 1 to 200 atoms and after the release of the specific biding member the remaining detectable group and the mobility modifier combined have a molecular weight of about 150 to 10,000 Daltons. A large number of specific label-mobility modifier pairs, each with a defined charge/mass ratio and therefore electrophoretic mobility, can be combined with various specific binding members to tag a large number of analytes (commercialized by Aclara, Mountain View, Calif.). U.S. Pat. No. 6,682,887 to Singh et al. discloses the use of electrophoretic tags for the analysis of specific nucleic acid sequences, wherein the detection sequence forming a specific binding pair is degraded to release the e-tag. In U.S. Pat. No. 6,686,152 Singh et al. disclose a method employing e-tags for detecting multiple nucleic acid sequences, wherein a nuclease cleavage releases the e-tags that are subsequently detected based on their unique electrophoretic mobility.
Another electrophoretic method, isoelectric focusing (IEF), has also been used for the detection of analytes. In isoelectric focusing, under the influence of an electric field, a molecule migrates in a medium of a defined pH gradient till it reaches a point where it carries no net charge, that is, till it reaches its isoelectric point (pI). At that point the pH of the medium is identical to the pI of the molecule. Isoelectric focusing is an equilibrium method, in contrast to electrophoresis performed in a medium without a pH gradient. In U.S. Pat. No. 5,348,633 Karger et al. disclose a capillary isoelectric focusing method for detecting analytes by the use of antibody Fab′ fragments labeled with a fluorescent dye, wherein the specific binding pairs and free antibody fragments are separated and detected. U.S. Pat. No. 5,376,249 to Afeyan et al. discloses a method of analysis utilizing isoelectric focusing wherein a detector is positioned at a predetermined spot along the capillary length corresponding to the isoelectric point of a binding pair formed between the analyte and an analyte-specific binding moiety.
In U.S. Pat. No. 5,824,478 Müller discloses a diagnostic method and probes wherein an analyte is contacted with a detector probe and a capture probe, with the detector probe including a moiety having a predetermined pI and a detectable label. Specificity of this method relies on the formation of a binding pair between the capture probe and the analyte. The analyte participates also in formation of another binding pair, with a detector probe. The moiety that defines a pI is released from the binding pair prior to its detection by isoelectric focusing. In a detector probe consisting of phycoerythrin-streptavidin, streptavidin served as a group specific (not analyte specific) binding member capable of interacting with all molecules containing a biotin group, while the highly fluorescent protein phycoerythrin was both the label and the pI determining moiety. As disclosed further in U.S. Pat. No. 5,824,478, a chemical treatment of phycoerythrin, or phycoerythrin-avidin complex, may result in production of modified proteins that have pI values in the pH range of 6-9. However, attempts to fractionate these modified proteins into useful fractions of defined pI values proved to be difficult, as subsequently described by Cruickshank et al. in Journal of Chromatography A 817:41-47, 1998. Instead, small synthetic peptides, from three to eight residues, labeled with a fluorescent dye were found to provide the required moieties of defined isoelectric points. The fluorescently labeled peptides had to be released from specific binding pairs prior to isoelectric focusing. While the use of small peptides represents an improvement, the method disclosed by Müller et al. and further described by Cruickshank et al. still suffers from drawbacks. Thus, the need for two probes, a detector probe and an analyte-specific capture probe, makes the method complex and expensive. The need to release the moiety that contains a label and determines the pI prior to isoelectric focusing increases complexity of the method and may result in undesirable side reactions, some of which were described in the Journal of Chromatography article. In addition, many biological samples contain proteases that could degrade the peptides serving as the analyte specific tags. Such protease degradation could lead to misinterpretation of results, because the signal from a degraded tag would be absent, or the signal would be lower in the case of partial degradation of the tag, or a degraded tag could be mistaken for another tag in the case that the degraded tag is of a similar or identical pI as one of the original tags. Finally, this method suffers from a low sensitivity of analyte detection, because it relies on the attachment of a single dye molecule to the peptide moiety having a defined pI. In U.S. Pat. No. 5,824,478 Müller mentioned a possibility of using a moiety having many fluorescent molecules, specifically, a dextran microparticle of several nanometers diameter, produced by Molecular Probes, Eugene, Oreg., wherein the microparticle would be linked to a detector probe by a polystyrene spacer-arm containing a cleavable bridge, such as a disulfide bond. It was not disclosed, however, how such a fluorescent dextran particle could become a moiety with a defined isoelectric point, the requirement necessary for functioning of the method of U.S. Pat. No. 5,824,478.
From the above description it is evident that there still exists a need for a method that could realize all the benefits of employing isoelectric focusing for analyte detection. Such a method, based on a novel isoelectric particle, is disclosed herein.